外源性人野生型p53基因转染导致兔动脉硬化斑块的不稳定性

目的　建立动脉粥样硬化不稳定性斑块的动物模型。方法　 6 4只雄性纯种新西兰大白兔随机分成A组 (5 4只 )与B组 (1 0只 ) ,A组用球囊损伤腹主动脉 高脂喂养 1 0周 ,B组仅给予高脂喂养 1 0周。于 8周末将A组随机分为A1和A2两个亚组 ,在腹主动脉斑块形成处分别转染携带人野生型 p5 3基因或LacZ基因的重组腺病毒载体 ,2周后A1 (p5 3基因 )和A2 (LacZ基因 )组各处死1 0只 ,观察斑块自发破裂情况。分别给予余下的兔中国斑点蝰蛇毒 (CRVV)和组胺药物触发斑块破裂 ,然后将兔处死取出腹主动脉进行病理学及免疫组化检查。实验开始及处死前进行血脂检查。结果　A1组转染 p5 3阳性细胞数比其他两组明显增加 (分别为 32 4 %± 1 0 2 %,1 5 8%± 3 6 %,1 6 2 %± 6 7%,P均 <0 0 0 1 ) ,细胞凋亡率明显高于其他两组 (分别为 2 5 %± 0 8%,1 0 %±0 3%,0 9%± 0 4 %,P均 <0 0 1 ) ,斑块纤维帽显著变薄 (P <0 0 5 ) ,血管平滑肌细胞减少 ,巨噬细胞聚集 ,药物触发后有 1 2只共 2 0处发生斑块破裂及血栓形成 ,A2组中 ,药物触发后仅 5只 7处发生斑块破裂。B组未见斑块破裂及血栓形成。结论　应用外源性人野生型p5 3基因转染动脉硬化兔可导致斑块的不稳定性 ,药物触发后出现斑块破裂及血栓形

Induction of atherosclerotic plaque instability in rabbits after transfection of human wild-type p53 gene

OBJECTIVE: To develop an animal model of unstable atherosclerotic plaques. METHODS: Sixty-four New Zealand white rabbits were randomly divided into two groups: group A (n = 54, undergoing balloon-induced abdominal aortic wall injury and then fed on a diet of 1% cholesterol) and group B (n = 10, fed on a diet of 1% cholesterol only). At the end of the eighth week, the rabbits in group A were randomly divided into two subgroups: group A1 (n = 27) and group A2 (n = 27). Recombinant adenovirus carrying human wild-type p53 gene and beta galactosidase (LacZ) genes were injected through a catheter into the aortic segments rich in plaques in group A1 and A2 respectively. Two weeks later, 10 rabbits each in group A1 and A2 were killed to observe the occurrence rate of spontaneous plaque rupture, and the remaining rabbits in group A1, A2 and B all underwent pharmacological triggering by Chinese Russell's viper venom injected subperitoneally and histamine injected intravenously two times with an interval of 24 hours. Twenty-four hours after the second pharmacological triggering the remaining rabbits were all killed and their abdominal aortae were taken out to undergo macropathologic observation, staining, and immunohistochemistry to examine the accumulation of macrophages and the expression of p53 protein, in the plaques with and without rupture respectively. At the beginning of the experiment and before being killed the serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein choloesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were examined. RESULTS: Before the rabbits were killed the blood-lipid levels were significantly increased in comparison with the baseline levels (all P < 0.01), however, without significant differences among the three groups. The rate of cells positive in p53 transfection was 32.4% +/- 10.2% in the group A1, significantly higher than those in the group A2 and group B (15.8% +/- 3.6% and 16.2% +/- 6.7% respectively, both P < 0.001). The fibrous cap of the plaque was significantly thinner in the group A1 than in the other 2 groups (both P < 0.05). The apoptosis rate was 2.5% +/- 0.8% in the group A1, significantly higher than those in the group A2 and group B (1.0% +/- 0.3% and 0.9% +/- 0.4% respective, both P < 0.01). The accumulation of macrophages within the plaques was significantly remarkable and the number of vessel smooth muscle cells was much smaller in the group A1. Plaque rupture and thrombosis occurred in 12 rabbits, numbering 20 lesions, after pharmacological triggering in the group A1, and occurred in only 5 rabbits, numbering 7 lesions, in the group A2. None of the rabbits in group B showed any lesions after the pharmacological triggering. CONCLUSION: With transfection of human wild-type p53 genes and pharmacological triggering, plaque rupture and thrombosis may occur in most atherosclerotic lesions in rabbits, which offered a reliable model for the further study of unstable plaques.